The proteins of the nuclear lamina have generated enormous interest because of recent studies showing that mutations in the gene for lamin A/C (LMNA) develop a host of different diseases, including cardiomyopathy, muscular dystrophy, and partial lipodystrophy. The objectives of this proposal are to define the enzymes that are important in the posttranslational processing of the nuclear lamins and to understand the consequences of defective posttranslational processing at both the cellular and tissue levels. Prelamin A (664 amino acids) terminates with a "CAAX" sequence motif and undergoes a complicated series of posttranslational modifications. First, the cysteine (C) of the CAAX motif is farnesylated by protein farnesyltransferase. Second, the last three amino acids of the protein (i.e., the -AAX) are released by a prenylprotein-specific endoprotease (likely Rce1 or Zmpste24 or both). Third, the newly exposed farnesylcysteine is methylated by isoprenylcysteine carboxyl methyltransferase (Icmt), a membrane protein of the endoplasmic reticulum. Fourth, once the cell has gone to all of this effort, the carboxyl-terminal 15 residues of the protein (including the farnesylcysteine methyl ester) are clipped off and degraded, leaving mature lamin A (646 amino acids). Zmpste24 might carry out that final endoproteolytic-processing step. Lamin B1 and B2 have a CAAX sequence motif and undergo the first three processing steps, but do not undergo a second endoproteolysis step; thus, their sequences terminate with a methylated farnesylcysteine. During the past few years, the laboratory of Dr. Stephen Young has generated knockout alleles [as well as some conditional ("floxed") alleles] for many of the genes involved in CAAX protein processing (e.g., Fntb, Rce1, Zmpste24, and Icmt) for the purpose of analyzing the importance of the posttranslational processing steps. In mice lacking Zmpste24, the processing of prelamin A to lamin A was blocked. Of note, the Zmpste24-deficient mice exhibited reduced muscle strength (suggestive of a laminopathy), and also developed spontaneous bone fractures, a peculiar finding not generally observed in humans with lamin mutations. The first aim of this grant application is to define, biochemically, the precise role(s) of Zmpste24 in prelamin A processing. The second aim is to further define the cellular and tissue pathology of Zmpste24 mice and then to determine whether all of the pathologic findings are due to defective prelamin A processing. The third aim is to understand the posttranslational processing of lamin B1 and to define the consequences of lamin B1 deficiency in mammals.